Proteome analysis of Saccharomyces cerevisiae after methyl methane sulfonate (MMS) treatment

Abstract

The treatment of methyl methane sulfonate (MMS) increases sensitivity to the DNA damage which, further leads to the cell death followed by a cell cycle delay. Delay in the cell cycle is because of the change in global transcription regulation which results into proteome change. There are several microarray studies on the transcriptome changes after MMS treatment, but very few studies are reported related to proteome change. The proteome analysis in this report identified subgroups of proteins, belonging to known cell cycle regulators, metabolic pathways and protein folding. About 53 proteins were identified by MS/MS and found that 36 of them were induced, 10 were repressed and few of them showed insignificant change. Our results indicated the change in the interactome as well as phosphorylation status of carboxy terminal domain (CTD) of RNA Polymerase II (RNAP-II) after MMS treatment. The RNAP-II complex was affinity purified and ~1640 peptides were identified using nano LC/MS corresponding to 27 interacting proteins along with the twelve RNAP-II subunit. These identified proteins participated in the repair of the damage, changes the function of the main energetic pathways and the carbon flux in various end products. The main metabolic enzymes in the glycolysis, pyruvate phosphate and amino acid biosynthesis pathways showed significant change. Our results indicate that DNA damage is somehow related to these pathways and is co-regulated simultaneously.

Keywords: DNA damage; Mass spectrometry (MS); Methyl methane sulfonate (MMS); Proteomics; RNA polymerase II-CTD.

Fig. 1

MMS sensitivity of yeast cells at different temperature. (A) The cultures were grown in YPD until the mid-log phase and a serial tenfold dilution were prepared and spotted onto YPAD plates containing different concentrations of MMS and incubated for two days. (B) Growth curve of yeast cell in the presence and absence of 0.02% MMS at 30 °C.

Fig. 2

Image of 2D gel (10% SDS-PAGE) labeled with the Spot ID and the name of the protein identified by MALDI. Red and black arrows represent the induced and repressed spots respectively, while orange arrow shows spot with no significant change in the expression. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Functional characterization of identifies proteins. (A) Diagrammatic representation of the identified proteins in 4 major metabolic pathways. (B) Average fold change in expression of the proteins related to a particular pathway (calculated by the average of fold change expression in total identified proteins in the specific pathway).

Fig. 4

CTD phosphorylation leads to change in the interactome of RNAP-II. (A) Cells were grown till 0.8 OD and treated with 0.02% MMS and the samples were collected at different time interval. Western blotting was done to check the Ser2, Ser5 and Ser7 phosphorylation status using 3E10, 3E8 and 4E12 antibody, respectively. Rpb1 level was checked using anti-TAP antibody. (B) Schematic representation of the strategies for the identification of the RNAP-II associated proteins. (C) 10% SDS-PAGE where Lane1 represent the ladder and lane2, 3 and 4 represent RNAP-II purification from JTY1, Y1056 and Y1088 respectively.

Fig. 5

Network mapping of the RNAPII associated protein using string 10. The nodes represent the protein. The empty node represents the protein without 3D structure while the filled node represents the protein with known or predicted 3D structure. The colorful lines are called edges which represents the protein-protein interaction. The edges show the number of evidence of protein-protein interactions. The network mapping shows the proteins related to RNA biogenesis and basal transcription factors.